Polysaccharide (PS)-encapsulated bacteria are a major cause of infections in humans. Anti-PS IgG antibodies (Ab) are critical to host defense against infection with these bacteria. The function of anti-PS Ab includes complement fixation and opsonization of bacteria for uptake and killing by phagocytes. The mammalian IgG anti-PS Ab response, however is restricted to only one or two subclasses (IgG3 in mice, IgG1 and IgG2 in man). Defining the relative function of anti-PS Ab of different IgG subclasses is crucial to better understanding of immunity to PS-coated bacteria as well as the pathogenesis of several human disease states such as IgG subclass deficiency, and chronic pneumonia in Cystic Fibrosis where inappropriate IgG subclass of anti-P. aeruginosa (PA) Ab may contribute to inability to clear PA from the lung. The relative function of anti-PS Ab of different IgG subclass remains unclear due to previous studies primarily using polyclonal affinity purified Ab exposed to chaotropic agents that alter Fc function, or to the use of monoclonal Ab with different antigenic specificities. In this proposal, we will precisely define the role of IgG subclass in anti-PS Ab effector function. First, we will make murine monoclonal Ab (MAb) directed to O- PS-side chain of PA LPS and to mucoid exopolysaccharide of PA. Next, using sequential sublining, we will make switch variants of these MAb that have identical variable regions but are of different IgG subclasses. The variable region genes will be cloned and ligated to the gamma constant region human genes to make human/murine chimeric transfectomas producing Ab of all four human IgG subclasses that have identical variable regions. The functional characteristics of the murine and chimeric anti-PS Ab will be compared in terms of: 1) ability to fix complement, 2) ability to opsonize PA for uptake by phagocytes, and 3) protective efficacy in animal models of PA infection. Finally, to determine the in vivo importance of the predominant anti-PS IgG subclass, IgG3 deficient mice will be mae by targeting the murine gamma 3 gene for mutation and homologous recombination in embryonic stem cells. These mice will be evaluated for their ability to immunologically respond to PS, PS-protein conjugates, and bacterial infection. These studies will determine which anti-PS IgG subclass functions most efficiently against PS-coated bacteria, better define the mechanism of functional differences between IgG subclasses and explore the immunologic relevance of these differences in vivo. This information will allow more rational strategies of active and passive immunization against PS-coated bacteria and improved treatment of IgG subclass deficiencies.